Based upon our study on the partitioning of HCV RNA during Cohn-Oncley fractionation of plasma, 50% of HCV RNA present in the starting plasma was found in cryoprecipitate, the source material for the manufacture of Antihemophilic Factor (Human) (AHF). We have used a similar PCR method (with primers from the 5' non-coding region of HCV) to examine levels of HCV RNA in more than 180 lots of AHF, including products produced with and without viral inactivation steps. All lots were submitted by 6 manufacturers for FDA release and have been stored at 4 degrees C. Each AHF vial was reconstituted to half of the volume specified by the manufacturer, and the sample aliquots were then digested with proteinase K in the presence of SDS at 60 degrees C for 1 h prior to RNA extraction and PCR. The sensitivity of the assay was determined by using H-strain plasma known to contain 106.5 chimpanzee infectious doses/ml. This sample was found to contain 6 x 106 PCR units of HCV RNA/ml which is comparable to the sensitivity of the chimpanzee model. All but one of 37 lots which had undergone no viral inactivation procedure and been manufactured before 1984 were positive for HCV RNA, with levels ranging from 0.2 to 189 PCR U/IU FVIII. All 15 lots of dry-heated AHF were positive for HCV RNA whereas 13 of 16 lots of wet-heated AHF were positive. Wet-heated AHF had relatively lower levels of HCV RNA than dry-heated AHF. Solvent/detergent (S/D) treatment was very efficient in eliminating HCV RNA from AHF prepared by 2 manufacturers. However 12 of 20 lots of S/D treated AHF produced by a third manufacturer did have detectable, albeit low, levels of HCV RNA (less than 0.2 PCR U/IU FVIII). All immunoaffinity purified AHF lots assayed were negative for HCV RNA. All 38 lots manufactured in 1992 and 1993 were negative. This may be attributed to the implementation of anti-HCV screening of plasma by manufacturers in 1992. Thus, even though a majority of HCV RNA in plasma partitions into cryoprecipitate, the current viral inactivation procedures in combination with anti-HCV screening of plasma efficiently eliminate HCV RNA from AHF preparations.